MNQ derivative D21 protects against LPS-induced inflammatory damage in bovine ovarian follicular GCs in vitro via the steroid biosynthesis signaling pathway

Abstract

Bacterial infections of the reproductive system of dairy cows lead to inflammation, and lipopolysaccharide (LPS) of the cell wall of Gram-negative bacteria is the main pathogenic component of inflammation. LPS inhibits follicular growth and development and alters the expression of follicular granulosa cells (GCs) genes in the ovary, leading to their functional disorders. Naphthoquinones have anti-inflammatory effects. In this experiment, 2-methoxy-1,4-naphthoquinone (MNQ), an extract of Impatiens balsamina L, and its derivative D₂₁ were used to eliminate the inflammatory response of GCs exposed to LPS in vitro and to restore functional disorders in GCs. The anti-inflammatory effects of the two compounds were compared and their mechanism of action was investigated. The cytotoxicity of MNQ and its derivative D₂₁ on follicular GCs was determined by MTT method. The relative expression of inflammatory factors and steroid synthesis-related genes were determined by qRT-PCR. The protective effects of MNQ and D₂₁ on cellular inflammatory damage were observed by TEM. ELISA were performed to detect the levels of estradiol (E₂) and progesterone (P₄) in the culture supernatant. The expression of differential genes was analyzed by RNA-seq, and GO and KEGG enrichment analysis of differential genes were performed to investigate the mechanism of anti-inflammatory effect of D₂₁. The results showed that the maximum no-cytotoxic concentrations of MNQ and D₂₁ acting on GCs for 12 h were 4 μM and 64 μM, respectively. LPS concentration of 10 μg/mL had little effect on the survival of follicular GCs, but the relative expressions of IL-6, IL-1β and TNF-α were significantly higher (P < 0.05). The results of qRT-PCR, ELISA and TEM observations showed that the anti-inflammatory effect of D₂₁ was stronger than that of MNQ. RNA-seq analysis revealed a total of 341 differential genes between the LPS vs CK group (Control group) and the D₂₁+L vs LPS group, which were mainly enriched in signaling pathways such as steroid biosynthesis. Nine genes in this signaling pathway were analyzed, and the RNA-seq and qRT-PCR results were found to be basically consistent. In this study, we confirmed that derivative D₂₁ has stronger in vitro anti-inflammatory effects and better efficacy in protecting bovine follicular GCs from inflammatory damage than MNQ and acts through the steroid biosynthesis signaling pathway.

Keywords: Derivative D(21); GCs; Inflammatory response; LPS; MNQ.